RU2530719C1 - Method for prediction of delayed puberty in prepubertal boys suffering obesity - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: enzyme immunoassay is used to determine blood serum leptin in the boys at the age of 11-12 years old; the body weight index (BWI) is calculated; and leptin is related to the derived BWI. If the calculated numerical value is more than 0.4, enzyme immunoassay is used to measure dihydrotestosterone (DHT) and anti-Mullerian hormone (AMH); if the derived relation value makes 30.86 and less, delayed puberty is predicted in the prepubertal boys.

EFFECT: higher accuracy.

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Description

The invention relates to medicine, namely to endocrinology, and will find use in predicting reproductive dysfunction in obese boys in the prepubertal period.

According to epidemiological studies in the Russian Federation, the prevalence of overweight in children in different regions of Russia ranges from 5.5 to 11.8%, 5.5% of children living in rural areas and 8.5% of children in cities are obese. (Peterkova V.A. and co-authors of 2004)

The study of the reproductive health of boys has recently become increasingly important due to the fact that the male factor in childless marriage is 40-60% and has a tendency to increase. The prerequisites of reproductive health disorders detected in adults can arise already in the early stages of ontogenesis - from the moment of formation of the zygote, to the very moment of the reproduction of offspring. As a result, untimely detection and correction of diseases of this type in children and adolescents can lead to extremely negative consequences.

Puberty is the period of human ontogenesis during which the final maturation of the hypothalamic-pituitary system occurs. The consequences of synchronization of endocrine phenomena in puberty are the development of secondary sexual characteristics and fertility, a change in metabolic rate and anthropometric characteristics - growth and body proportions (MeSH).

Functional disorders of the reproductive system, beginning in childhood and adolescence, are studied today in the genetic, endocrine and metabolic aspects (Dedov, Melnichenko, Chebotnikova, 2006). Currently, the main diagnostic algorithm aimed at identifying and differential diagnosis of various diseases manifesting as delayed sexual development in boys is the one proposed in the “Endocrinological National Guide” (edited by Dedov, Melnichenko 2009). In accordance with this approach, an anamnesis is studied, a physical examination, an assessment of sexual development according to Tanner and a hormonal study are carried out, including the determination of basal levels of gonadotropic (luteinizing (LH) and follicle-stimulating (FSH)) and sex hormones (primarily testosterone).

In addition, it is necessary to conduct a standard test with the release factor of luteinizing hormone, after intravenous administration of which is determined after 24 hours LH FSH and Testosterone. With a functional delay in sexual development, LH and testosterone increase to puberty. However, this test is informative if the patient’s bone age is at least 12-13 years, which lags behind with a functional delay in sexual development, as a result, the reaction to the stimulation test may be absent. Therefore, a diagnostic study can be started at 13-14 years old, when the growth retardation of the muscular skeleton, the absence of voice mutation, the absence of secondary sexual characteristics and the stage of sexual development are already expressed according to Tanner 1. Such late detection of even a functional delay in sexual development, not to mention already about hypogonadotropic hypogonadism, leads to psychosocial deprivation of a teenager and can forever leave an imprint on his entire subsequent life and development.

Due to the need for early preventive diagnosis of reproductive dysfunctions and the limitations of the standard algorithm, we have developed a methodology for identifying predictors of reproductive disorders that lead to delayed sexual development due to metabolic disorders in boys who are obese in prepubertal (11-12 years old).

The objective of the invention is the development of an effective method for predicting delayed sexual development in boys who are obese in prepubertal, which will determine the effectiveness of the therapy.

The problem is solved in that for boys of prepubertal age (11-12 years), BMI is determined for obesity (weight in kg divided by m ² growth), a blood level of leptin is examined and the leptin / BMI ratio is calculated. If the numerical value of this ratio is 0.4 or more, the dehydrotestosterone (DHTS) levels of the anti-Muller hormone (AMH) are additionally determined by ELISA, their ratio is calculated, and if its numerical value is less than 30.86, the delayed sexual development of the subject in the pubertal period is predicted (13-14 years old).

This method is characterized by low invasiveness and the diagnosis of metabolic disorders in the form of leptin resistance and allows us to assess the risk of delayed sexual development in puberty in 11-12 year old boys. A new technical result obtained by using the proposed method shows the high sensitivity and efficiency of the method, allows you to obtain objective data in a single study.

The main mediator of the endocrine interaction of adipose tissue and the reproductive system is the leptin signaling system. Leptin acts on the centers of hunger and satiety in the hypothalamus, participates in the brain regulation of energy homeostasis and controls body weight by reducing the synthesis and release of the neuropeptide that causes hunger. The role of leptin and the nature of its regulation in humans continue to be clarified, but it is already known that dysregulation of its secretion and leptin resistance are considered as one of the leading mechanisms for the development of obesity. Leptin and its receptors in the body are tissue-specific for Leydig cells, testicular tubules, epididymis, as well as in spermatozoa (compartmentalization in the acrosome). These data together suggest the involvement of the leptin system in the modulation of the processes of gametogenesis and androgenesis. This idea is confirmed there that with an increase in the amount of fat, the male body undergoes feminization, since it increases the levels of estrogen and leptin, which directly inhibits the production of testosterone.

In our study, hyperleptinemia was observed in obese children in both prepubertal and puberty (median leptin 20.0 pg / ml and 13.9 pg / ml) and significantly differed from children in the control group (median leptin 5.04 pg / ml and 3.36 pg / ml, respectively). The level of leptin significantly correlated with the severity of obesity and biochemical parameters, namely, BMI (r = 0.79; p≤0.05), insulin resistance index (r = 0.61) and insulin (r = 0.75).

We noted that not all obese children had a delay in sexual development, in connection with which we began to consider the relationship of leptin with BMI. When revealing the ratio of leptin / BMI of 0.4 and higher, a greater number of subjects had a delay in sexual development.

Traditional blood tests for testosterone, LH and FSH, usually performed as a standard for diagnostic tests, cannot predict at 11-12 years of age which boys with obesity will have a delayed sexual development of 14 years.

A special place in the functioning of the reproductive system is occupied by AMG. According to the literature, AMG reflects the number and quality of Sertoli cells in boys before puberty, their determination is possible in assessing male fertility at any age, starting from the neonatal period. An increase in the level of AMH in prepubertal is associated with a decreased concentration of androgen in the blood serum - TS and DHTS. With puberty, AMH levels decrease, reflecting the maturation of Sertoli cells in response to the action of androgens. AMH confirms approaching puberty more reliably than more variable testosterone and LH.

Our studies of obese children of prepubertal 11-12 years old with sexual development Tanner 1 did not reveal significant differences among the level of antimüller hormone from children with normal weight and the initial phase of sexual development (median AMH 60.20 ng / ml and 79, 20 ng / ml, respectively). Whereas between the 1st and 2nd stage of sexual development in boys 11-12 years old with obesity there are significant differences (p <0.005). In this regard, AMH alone cannot be a specific diagnostic marker.

Therefore, having examined the correlation relationships and the ratio of AMH with sex hormones (testosterone, LH, FSH, DHTS), we found the pattern that interests us only in connection with DHTS.

This is due to the fact that DHTS is more closely associated with the androgen intracellular receptor than testosterone, so its performance is more stable and can be used in diagnosis. However, the scatter of its indicators is large and it cannot act as a specific diagnostic marker in itself. Using the ROC analysis, we identified diagnostic criteria for the DHTS / AMH ratio.

A detailed description of the method and examples of its clinical use.

All boys with obesity 11-12 years old determine the height, weight and evaluate the stage of sexual development according to Tanner. In all children, BMI is determined by the ratio of weight to height (meter squared). Next, a blood sampling is performed to determine hormones in the blood, including leptin.

Blood sampling is carried out in the morning (from 8.00 to 11.00) in compliance with the established requirements, using the vacuum method. Blood serum is obtained by centrifugation for 10 min at 3000 rpm. Prior to the study, blood serum is stored in a freezer at a temperature of -20 ° C.

In the blood, the level of leptin is determined and, with its ratio to BMI of 0.4 or more, a blood test is performed on DHTS and AMH.

Studies of these hormones are carried out by enzyme-linked immunosorbent assay on an automatic enzyme-linked immunosorbent analyzer "Alisei" (Italy) using test systems manufactured by "Alkor Bio" (Russia), DRG (Germany, USA).

The principle of the method for determining the level of leptin is to use a “sandwich” variant of enzyme-linked immunosorbent assay. Highly specific monoclonal antibodies are used in the kit: monoclonal antibodies specific for leptin are immobilized in the wells of a microplate, and other monoclonal antibodies specific for another leptin epitope are conjugated to biotin. At the first stage of the analysis, the leptin contained in the samples and standards binds to immobilized antibodies and biotinylated antibodies, forming a “sandwich” complex. Excess and unbound biotinylated antibodies are removed at the washing stage.

At the second stage of the analysis, the horseradish streptavidin peroxidase conjugate is added, which specifically binds to biotinylated antibodies. Then, unbound conjugate is removed by washing. After this, a substrate (IML) is introduced, which, as a result of the enzymatic reaction, forms a blue product, and the color is directly proportional to the amount of leptin present. The enzymatic reaction is stopped by the addition of a stop reagent, converting blue to yellow. The optical density of the solution is measured on a photometer at a wavelength of 450 nm, which is necessary to prepare a standard curve from which the amount of leptin in the samples can be directly read.

Analysis progress

1. All reagents were warmed to room temperature before use.

2. Prepared working solutions of streptavidin / HRP conjugate and wash buffer.

3. Pipette 20 μl of each standard, control and duplicate sample into appropriately labeled wells.

4. 80 μl of a solution of monoclonal antibodies to leptin conjugated with biotin were added to all wells.

5. Incubated for 1 hour at room temperature on a shaker (approximately 200 rpm).

9. The cells were washed 3 times, using 300 μl of diluted wash buffer per well, and then tapped with a microplate on filter paper, making sure that the wells were dry.

10. 100 μl streptavidin / HRP conjugate solution was added to each well.

11. Incubated on a shaker for 30 minutes at room temperature (approximately 200 rpm).

12. Washed the wells as indicated in paragraph 5.

13. 100 μl of TMB substrate was added to each well.

14. Incubated on a shaker for 10 minutes at room temperature.

15. Contributed 50 μl of stop solution to all wells, in the same sequence and at the same speed as in step 13.

16. The optical density of the solution in the wells was measured at a wavelength of 450 nm.

17. After measuring the optical density of the solution in the wells, based on the calibration graph, the concentration of the hormone in the determined samples was calculated.

In the study of dehydrotestosterone, a “competitive” version of enzyme-linked immunosorbent assay was used. The principle of the method is as follows: unlabeled antigen (dehydrotestosterone present in samples, controls and standards) and enzyme labeled antigen (conjugate) during incubation compete for a limited number of antibody binding sites immobilized in the wells of the microplate. Then, after washing, an enzyme substrate is added. The enzymatic reaction is stopped by the addition of a stop solution. The degree of staining formed during the enzymatic reaction is inversely proportional to the concentration of the hormone in the sample. The intensity of the developed staining is determined by measuring the absorption in the wells at a wavelength of 450 nm, using two comparison wavelengths in the range of 600-630 nm and 405 nm. After measuring the optical density of the solution in the wells, the concentration of the hormone in the determined samples is calculated based on the calibration graph.

Determination of dehydrotestosterone

Analysis progress

1. All reagents were mixed before analysis and brought to room temperature.

2. 100 μl of conjugate solution were added to all wells.

3. 50 μl of calibration samples, control and test serum were added to the corresponding wells.

4. The strips were incubated for 1 hour at room temperature with shaking on a shaker at a speed of 200 rpm.

5. At the end of the incubation, the contents of the wells were removed by decantation and the wells were washed 3 times.

6. 150 μl of TMB solution was added to all wells and the strips were incubated in the dark at room temperature (+ 18 ... 25 ° C) for 15-30 minutes depending on the degree of color development.

7. Added 50 μl stop reagent to all wells to stop the enzyme reaction, shake on a shaker for 1-2 minutes.

8. The optical density of the solution was measured at a wavelength of 450 nm.

9. The concentration of the hormone in the determined samples was calculated based on the calibration graph.

When studying the anti-Muller hormone in the AMH Gen II kits, an enzymatically enhanced sandwich variant of enzyme-linked immunosorbent assay was used. During production, standards, controls and samples are incubated in the wells of a microplate coated with antibodies to AMH. After incubation and washing, anti-AMH detecting antibodies conjugated with biotin are added to the wells of the microplate. After the second incubation and washing, the streptavidin-peroxidase conjugate (streptavidin-HRP) is added to the wells. After a third incubation and washing in the wells, a substrate of tetramethylbenzidine (TMB) is introduced. At the last stage of the procedure, a stop solution (acid) is added to the wells. The intensity of the developed staining is determined by measuring the absorption in the wells at a wavelength of 450 nm, using a comparison wavelength in the range of 600-630 nm. The measurement of absorption is directly proportional to the concentration of the determined hormone in the samples. After measuring the optical density of the solution in the wells, the concentration of the hormone in the determined samples is calculated based on the calibration graph.

Determination of anti-Muller hormone

Analysis progress

1. All reagents were mixed before analysis and brought to room temperature.

2. 20 μl of calibration samples, control and test serum were added to the corresponding wells.

3. Add 100 μl AMH Gen II buffer to each well.

4. Strips were incubated for 1 hour at room temperature with shaking on a shaker at a speed of 600-800 rpm.

5. At the end of the incubation, the contents of the wells were removed by decantation and the wells were washed 5 times.

6. 100 μl conjugate was added to all wells.

7. The strips were incubated for 1 h at room temperature and shaken on a shaker at a speed of 600-800 rpm.

8. At the end of the incubation, the contents of the wells were removed by decantation and the wells were washed 5 times.

9. Added 100 μl of streptavidin-enzyme conjugate to each well.

10. Strips were incubated for 30 minutes at room temperature and shaken on a shaker at a speed of 600-800 rpm.

11. At the end of the incubation, the contents of the wells were removed by decantation and the wells were washed 5 times.

12. 100 μl of TMB solution was added to all wells and the strips were incubated in the dark at room temperature for 10 minutes.

13. Add 100 μl stop reagent to all wells to stop the enzyme reaction.

14. The optical density of the solution was measured at a wavelength of 450 nm.

15. The concentration of the hormone in the determined samples was calculated based on the calibration graph.

The ratio of DHTS to AMH is calculated, and if the numerical value of this ratio is 30.86 or lower, the reproductive function in this boy is predicted in the pubertal period of development (13-14 years).

The performance of the proposed method is confirmed by the following clinical examples.

Example No. 1.

Patient T-ko Vadim, 11 years 6 months. When examining at the outpatient clinic - complaints of increased appetite and excess weight. An objective examination: the correct physique, increased nutrition with the predominant deposition of fat in the abdominal region. BMI is 23.53 kg / m ² . Diagnosed with obesity of 2 degrees. The skin is clean, no striae have been detected, the thyroid gland is not palpable. Constipation does not suffer. The genitals are developed according to the male type, the sexual formula is A1P1G1. Laboratory studies were performed: sex hormones LH = 1.32 mIU / ml, FSH = 1.9 mIU / ml, testosterone = 3.97 nmol / L, so the level of hormones corresponds to pre-pubertal values. Additionally, we determined the level of leptin = 10.46 ng / ml, and the ratio of leptin / BMI = 0.44 was calculated, which indicates the development of leptin resistance. In this connection, we further investigated the levels of AMH (18.8 ng / ml) and DHTS (280.62 pg / ml). Although the AMG indicators are not as high as in prepubertal, however, low DHTS for this age, in comparison with the control, indicate a delay in the response of sex hormones, therefore it is the ratio of DHTS / AMG = 14.93 that can serve as a more accurate diagnostic criterion for predicting delayed sexual development because suggests the risk of delayed sexual development in puberty. From a further diagnostic test with hCG and the appointment of stimulating therapy, the boy's parents refused.

The patient was assigned a sub-caloric diet, physical therapy exercises, dynamic observation. At the follow-up appointment after 6 months, it turned out that the parents did not comply with the recommendations on the teenager’s diet, at the age of 13 years and 10 months old complaints of overweight and stunting were revealed. Diagnosed with delayed sexual development.

Example No. 2.

Patient B-s Maxim, 12 years old. Complaints of stunted growth and sexual development. An objective examination: hypersthenic physique, increased nutrition with predominant deposition of fat in the abdominal region. Diagnosed with obesity of 2 degrees. BMI is 23.92 kg / m ² . The skin is clean, no striae have been detected, the thyroid gland is not palpable. Constipation does not suffer. The genitals are developed according to the male type, the sexual formula is A1P1G1. Laboratory studies were performed: sex hormones LH = 0.78 mIU / ml, FSH = 0.81 mIU / ml, testosterone = 1.41 nmol / L, so the level of hormones corresponds to pre-pubertal values. Biochemical studies with the determination of fat, carbohydrate and lipid metabolism, as well as the level of leptin (10.89 ng / ml), and the ratio of leptin / BMI equal to 0.46, which indicates the development of leptin resistance, were calculated. In this connection, we additionally determined the levels of DHTS and AMH, and calculated their DHTS / AMH ratio, which amounted to 30.86, which indicates a possible delay in sexual development in the puberty period.

The patient was assigned a sub-calorie diet, physical therapy exercises, dynamic observation. A stimulation test was performed with hCG with a positive response. In this connection, in a standard dose it is prescribed in order to stimulate the sexual development of hCG. After 6 months, a teenager has a weight loss, an increase in height, and a change in the sexual formula for A2P3G2. This example shows the effectiveness of the use of the proposed method for timely diagnosis and the initiation of stimulating therapy.

Example No. 3.

Patient M. Kurban, 11 years old, 8 months old, received complaints about overweight. An objective examination revealed a hypersthenic physique, an increased nutritional status with a uniform distribution of the subcutaneous fat layer. Diagnosed with obesity of 2 degrees. BMI is 24.24 kg / m ² . The skin is clean, no striae have been detected, the thyroid gland is not palpable. Constipation does not suffer. The genitals are developed according to the male type, the sexual formula is A1P2G2. Laboratory studies were performed: sex hormones LH = 2.86 mIU / ml, FSH = 3.16 mIU / ml, testosterone = 11, 67 ng / ml. Since the boy is obese, for prognostic purposes, we determined the level of leptin (10.57 ng / ml) and calculated the ratio of Leptin / BMI equal to 0.44, which indicates the development of leptin resistance. Additionally examined the performance of DHTS and AMG and calculated their ratio DHTS / AMG = 35.22. In this case, the studied indicators indicate that there is no threat of delayed sexual development in the puberty of the subject and does not require a diagnostic test with hCG and the appointment of stimulating hormonal therapy. The patient was assigned a sub-caloric diet, physical therapy exercises, dynamic observation.

The inventive method has been tested on a large clinical material. 70 students of 11-12 years old in five districts of Rostov-on-Don took part in our survey. The studies were carried out on the basis of the Research Institute of Biology of SFU. All boys examined were examined by endocrinologists at the Research Institute of Obstetrics and Pediatrics. All studies were conducted in accordance with international bioethical standards. Parents of participants in the study were informed about the goals and risks of the study and in writing confirmed their consent for their children to participate in the study.

The survey was conducted according to a single algorithm. The exclusion criteria from the examination were the presence of genetic diseases, syndromes and organic lesions of the central nervous system. To assess excess body weight and the degree of obesity, a body mass index (BMI) was calculated, SDS BMI corresponding to age and gender and a classification of body weight according to Knyazev Yu.A. were determined. (1982). Evaluation of sexual development was carried out on the basis of visual inspection and classification of stages of sexual development using the Tanner scale (Y. Tanner 1996). Hormonal studies included determination of serum: luteinizing hormone (LH), follicle-stimulating hormone (FSH), Testosterone (TS), anti-Muller hormone (AMH), Inhibin-B, dehydrotestosterone (DHTS), prolactin, androstenedione.

An increase in the level of AMH in the prepubertal period is associated with a reduced concentration of dehydrotestosterone (DHTS), and it is the ratio of DHTS / AMH that more fully reflects the functional state of the reproductive organs in boys and indirectly indicates the onset of puberty, when pulsed secretion of LH stimulates the formation of androgens in Leydig cells. Early identification of children at risk and the timely start of treatment will prevent the formation of further reproductive disorders. In the initial stages, measures aimed at combating BMI come to the forefront. This is the development of motivation, regulation and correction of eating habits, optimization of motor mode.

Statistical processing of the research results included the following methods: analysis of biochemical parameters was performed using the licensed STATISTICA version 6.0 package. Statistical data processing was performed using t-student criterion. The differences were considered statistically significant at p <0.05.

ROC characteristics of the criteria:

- leptin / BMI - sensitivity 82%, specificity 84%, AUC 0.833 (p = 0.0005);

- DHTS / AMH - sensitivity 76%, specificity 69%, AUC 0.738 (p = 0.0125).

The advantages of the proposed method are that

- it has rather high indicators of sensitivity and specificity, which makes this method informative and allows one to judge the presence of leptin resistance, even with a second degree of obesity;

- the ratio of DHTS / AMH in children with obesity 11-12 years allows us to predict the formation of delayed sexual development in the early stages;

- indicators DHTS / AMH in children with obesity 11-12 years can be further used as a criterion for the effectiveness of the therapy;

- There are no restrictions characteristic of the standard diagnostic algorithm.

Thus, the claimed method for predicting delayed sexual development in boys of prepubertal age with obesity will facilitate the timely adoption of measures aimed at safely stabilizing body functions and avoiding the development of reproductive disorders and problems of social adaptation in later life.

Claims (1)

A method for predicting delayed puberty in boys of prepubertal age with obesity by a blood test, characterized in that for boys aged 11-12, the serum leptin is determined by ELISA, the body mass index (BMI) and the ratio of leptin to BMI are calculated, and if it is 0.4 or more, in addition, the ELISA method determines the levels of dehydrotestosterone (DHTS) and anti-Muller hormone (AMH), and if their ratio is 30.86 or less, delayed sexual development in boys in the pubertal period is predicted.